In Vitro Biowaiver/
In Vitro
Bioequivalence
Platform

In vitro Biowaiver studies replace clinical BA-BE studies to classify the equivalence of two pharmaceutical products for approval. In vitro Biowaiver studies were initially applied for scale up and post approval changes (SUPAC) of generic product and later extended to approval of orally administered BCS class I (high solubility and high permeability) and class III (high solubility and low permeability) IR formulations.

In vitro Biowaiver studies involves characterizing comparative dissolution of test and innovator formulation, and gastrointestinal stability, solubility and Caco-2 permeability of active pharmaceutical ingredient (API).

Dissolution

A drug product (BCS class I) is considered RAPIDLY DISSOLVING when > 85% of the labelled amount of drug substance dissolves within 30 minutes in three different media (pH 1.2, 4.5 and 6.8) in a paddle (50 rpm) or basket (100 rpm) apparatus at 37°C in a volume of 900 mL.

A drug product (BCS class III) is considered ULTRA RAPIDLY DISSOLVING when > 85% of the labelled amount of drug substance dissolves within 15 minutes in three different media (pH 1.2, 4.5 and 6.8) in a paddle (50 rpm) or basket (100 rpm) apparatus at 37°C in a volume of 900 mL.

Solubility

The aqueous solubility of the drug is considered to be high when the highest single unit dose is COMPLETELY SOLUBLE in 250 mL or less of aqueous solution at (pH 1.2, 4.5 and 6.8) at 37°C.

Permeability

A drug substance is considered HIGHLY PERMEABLE when the extent of absorption in humans is determined to be > 85% of an administered dose, based on a mass balance determination or in comparison to an intravenous reference dose, in the absence of evidence suggesting instability in the gastrointestinal tract. Intestinal permeability is assessed by validated in vitro permeability models such as Caco-2.

• Method development and validation of test compound using HPLC and/or LC-MS/MS as per USFDA published Analytical/Bioanalytical guidelines
• Conducting dissolution, gastrointestinal stability and solubility of IR formulation/API in biorelevant media as per US pharmacopeia and analyzing the samples using validated HPLC/LC-MS/MS methods
• Assessing test compound in vitro Caco-2 permeability along with appropriate controls (high permeability, low permeability and efflux marker compounds) and analyzing the samples using validated LC-MS/MS methods
• Validation of Caco-2 permeability model with at least 20 model drugs binned in four different BCS classes
• Characterization of Caco-2 cell line with respect to the expression of various drug transporters

In vitro binding studies:

Locally acting compounds such as bile acid binders, phosphate binders and anti-ulcer drug, sucralfate (suspension/tablet) get released in the gastrointestinal environment which are not absorbed into the systemic circulation, binds with phosphate and bile acids to form insoluble complexes in the intestine and excreted through the faeces. Conventional clinical BA/BE studies do not allow to characterize the bioequivalence of locally acting drugs. Hence FDA has developed a set of In vitro BE guidelines for bile acid and phosphate binding drugs to compare the rate and extent of binding between Test and Reference formulations for bioequivalence establishment.

Kinetic binding study:

To characterize the maximum (saturation) binding of bile acids or phosphate to the test and innovator formulation with respect to time. This study is a support to the pivotal equilibrium binding study.

Equilibrium binding study:

The equilibrium binding study characterize Langmuir’s binding constants such as affinity (K1) and capacity constant (K2) for test and reference formulations. Bioequivalence is established between Test and Reference formulation based on K2 with an appropriate confidence interval.

• Method development and validation of bile acids/phosphate/Human serum albumin (HSA) or Bovine serum albumin (BSA) using HPLC/IC/ICP-MS as per USFDA published Bioanalytical guidelines
• Kinetic binding study is conducted for test and reference formulations with bile acids/phosphate/HSA or BSA concentrations and pH variation resembling the gastrointestinal pH. Free bile acids/phosphate/HSA or BSA concentration is determined and bound concentration is computed. Saturation binding with respect to time and binding similarity between test and reference formulation is characterized
• Equilibrium binding study for test and reference formulations with appropriate bile acids/phosphate ranging 8 different concentrations and pH variation resembling the gastrointestinal pH. Free bile acids/phosphate//HSA or BSA concentration is determined and bound concentration is computed to calculate Langmuir binding affinity (K1) and capacity (K2) constants
• Bioequivalence between test and reference formulation is determined based on the capacity constant (K2)